There is a need for an efficient system for introducing nucleic acid into live cells particularly in gene therapy. Genes are introduced into cells in order to achieve in vivo synthesis of therapeutically effective genetic products, e.g. in order to replace the defective gene in the case of a genetic defect. "Conventional" gene therapy is based on the principle of achieving a lasting cure by a single treatment. However, there is also a need for methods of treatment in which the therapeutically effective DNA (or mRNA) is administered like a drug ("gene therapeutic agent") once or repeatedly as necessary. Examples of genetically caused diseases in which gene therapy represents a promising approach are hemophilia, beta-thalassaemia and "Severe Combined Immune Deficiency" (SCID), a syndrome caused by the genetically induced absence of the enzyme adenosine deaminase. Other possible applications are in immune regulation, in which humoral or intracellular immunity is achieved by the administration of functional nucleic acid which codes for a secreted protein antigen or for a non-secreted protein antigen, by immunization. Other examples of genetic defects in which a nucleic acid which codes for the defective gene can be administered, e.g. in a form individually tailored to the particular requirement, include muscular dystrophy (dystrophin gene), cystic fibrosis (cystic fibrosis transmembrane conductance regulator gene), hypercholesterolemia (LDL receptor gene). Gene-therapy methods of treatment are also potentially of use when hormones, growth factors or proteins with a cytotoxic or immune-modulating activity are to be synthesized in the body.
Gene therapy also appears promising for the treatment of cancer by administering so-called "cancer vaccines." In order to increase the immunogenicity of tumor cells, they are altered to render them either more antigenic or to make them produce certain cytokines in order to trigger an immune response. This is accomplished by transfecting the cells with DNA coding for a cytokine, e.g. IL-2, IL-4, IFN gamma, or TNF alpha. To date, gene transfer into autologous tumor cells has been accomplished via retroviral vectors.
The mode of activity of antisense RNAs and DNAs as well as ribozymes enables them to be used as therapeutic agents for blocking the expression of certain genes (such as deregulated oncogenes or vital genes) in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells and exert their inhibiting effect therein (Zamecnik et al., 1986), even if their intracellular concentration is low, caused, inter alia, by their restricted uptake by the cell membrane as a result of the strong negative charge of the nucleic acids. The oligonucleotides may be modified, e.g. by substituting the charged phosphodiester groups by uncharged groups. Another possible method of direct modification consists in using nucleoside analogues.
Various techniques are known for gene transfer into mammalian cells in vitro but their use in vivo is limited (these include the introduction of DNA by means of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran or the calcium phosphate precipitation method).
In recent times, biological vectors have been developed to bring about the transfer of genes by using the efficient entry mechanisms of their parent viruses. This strategy was used in the construction of recombinant retroviral and adenoviral vectors in order to achieve a highly efficient gene transfer in vitro and in vivo (Berkner, 1988). For all their efficiency, these vectors are subject to restrictions in terms of the size and construction of the DNA which is transferred. Furthermore, these agents constitute safety risks in view of the co-transfer of viable viral gene elements of the original virus. Thus, for example, the use of retroviruses is problematic because it involves, at least to a small percentage, the danger of side effects such as infection with the virus (by recombination with endogenous viruses and possible subsequent mutation into the pathogenic form) or the formation of cancer. Moreover, the stable transformation of the somatic cells of the patient, as achieved by means of retroviruses, is not desirable in each case because this can only make the treatment more difficult to reverse, e.g. if side effects occur.
In order to circumvent these restrictions, alternative strategies for gene transfer have been developed, based on mechanisms which the cell uses for the transfer of macromolecules. One example of this is the transfer of genes into the cell via the extremely efficient route of receptor-mediated endocytosis (Wu and Wu, 1987, Wagner et al., 1990 and EP-A1 0388 758). This approach uses bifunctional molecular conjugates which have a DNA binding domain and a domain with specificity for a cell surface receptor (Wu and Wu, 1987, Wagner et al., 1990). If the recognition domain (hereinafter referred to as the "internalizing factor") is recognized by the cell surface receptor, the conjugate is internalized by the route of receptor-mediated endocytosis, in which the DNA bound to the conjugate is also transferred. Using this method, it was possible to achieve gene transfer rates at least as good as those achieved with the conventional methods (Zenke et al., 1990).
Whereas this vector system is able to transport large quantities of DNA into cells having the suitable cell surface receptor, the corresponding gene expression very often does not accord with the transfer capacity (Cotten et al., 1990). It was assumed, inter alia, that the reason for this phenomenon is that the DNA conveyed into the cell by receptor-mediated endocytosis lands in lysosomes where it undergoes degradation (Zenke et al., 1990, Cotten et al., 1990). Therefore, the fact that the DNA internalized in lysosomes does not have any specific mechanism for leaving the intracellular vesicle system constitutes a restriction which is inherent in this transport system.
The aim of the present invention was to reduce or eliminate these restrictions.
A plurality of viruses effect their entry into the eucaryotic host by means of mechanisms which correspond in principle to the mechanism of receptor-mediated endocytosis. Virus infection based on this mechanism generally begins with the binding of virus particles to receptors on the cell membrane. After this, the virus is internalized into the cell. This internalizing process follows a common route, corresponding to the entrance of physiological ligands or macromolecules into the cell: first of all, the receptors on the cell surface arrange themselves in groups, and the membrane is inverted inwardly and forms a vesicle surrounded by a coating. After this vesicle has rid itself of its clathrin coat, acidification takes place inside it by means of a proton pump located in the membrane. This triggers the release of the virus from the endosome. Depending on whether the virus has a lipid coat or not, two types of virus release from the endosome were taken into account: in the case of so-called "naked" viruses (e.g. adenovirus, poliovirus, rhinovirus) it was suggested that the low pH causes changes in configuration in virus proteins. This exposes hydrophobic domains which are not accessible at the physiological pH. These domains thus acquire the ability to interact with the endosome membrane and thereby cause the release of the virus genome from the endosome into the cytoplasm. As for viruses with a coat (e.g. vesicular stomatitis virus, Semliki Forest virus, influenza virus) it is presumed that the low pH modifies the structure or configuration of some virus proteins, thereby promoting the fusion of the virus membrane with the endosome membrane. Viruses which penetrate into the cell by means of this mechanism have certain molecular peculiarities which enable them to break up the endosome membrane in order to gain entry into the cytoplasm.
Other viruses, e.g. the coated viruses Sendai, HIV and some strains of Moloney leukaemia virus, or the uncoated viruses SV40 and polyoma, do not need a low pH for penetration into the cell; they can either bring about fusion with the membrane directly on the surface of the cell (Sendai virus, possibly HIV) or they are capable of triggering mechanisms for breaking up the cell membrane or passing through it. It is assumed that the viruses which are independent of pH are also capable of using the endocytosis route (McClure et al., 1990).
In experiments which preceded the present invention it was established that gene transfer by means of nucleic acid complexes in which the nucleic acid is complexed with polycations, optionally coupled to an internalizing factor, e.g. with transferrin-polylysine conjugates, is significantly increased by treatment with adenoviruses, specific retroviruses or with virus fragments. This effect was achieved by making use of the phenomenon that these viruses are taken up into the cells by endocytosis mechanisms and have a specific mechanism for escaping from the vesicle system by breaking open the endosomes, e.g. in the case of the adenoviruses (Pastan et al., 1986).
Starting from these observations, the problem of the invention was solved by developing a bioconjugate which contains the virus as an integral part of its functional construct.